This invention is directed to assays for measuring the activity of ubitquitination enzymes. The invention is also directed to assays for identifying modulators of ubiquitination.
Ubiquitin is a highly conserved 76 amino acid protein expressed in all eukaryotic cells. The levels of many intracellular proteins are regulated by a ubiquitin-dependent proteolytic process. This process involves the covalent ligation of ubiquitin to a target protein, resulting in a poly-ubiquitinated target protein which is rapidly detected and degraded by the 26S proteasome.
The ubiquitination of these proteins is mediated by a cascade of enzymatic activity. Ubiquitin is first activated in an ATP-dependent manner by a ubiquitin activating enzyme (E1). The C-terminus of a ubiquitin forms a high energy thiolester bond with E1. The ubiquitin is then passed to a ubiquitin conjugating enzyme (E2; also called ubiquitin carrier protein), also linked to this second enzyme via a thiolester bond. The ubiquitin is finally linked to its target protein to form a terminal isopeptide bond under the guidance of a ubiquitin ligase (E3). In this process, chains of ubiquitin are formed on the target protein, each covalently ligated to the next through the activity of E3.
The components of the ubiquitin ligation cascade have received considerable attention (for a review, see Weissman, Nature Reviews 2:169-178 (2001)). E1 and E2 are structurally related and well characterized enzymes. There are several species of E2 (at least 25 in mammals), some of which act in preferred pairs with specific E3 enzymes to confer specificity for different target proteins. While the nomenclature for E2 is not standardized across species, investigators in the field have addressed this issue and the skilled artisan can readily identify various E2 proteins, as well as species homologues (See Haas and Siepmann, FASEB J 11: 1257-1268 (1997)).
E3 enzymes contain two separate activities: a ubiquitin ligase activity to conjugate ubiquitin to substrates and form polyubiquitin chains via isopeptide bonds, and a targeting activity to physically bring the ligase and substrate together. Substrate specificity of different E3 enzymes is the major determinant in the selectivity of the ubiquitin-dependent protein degradation process.
Some E3 ubiquitin ligases are known to have a single subunit responsible for the ligase activity. Such E3 ligases that have been characterized include the HECT (homologous to E6-AP carboxy terminus) domain proteins, represented by the mammalian E6AP-E6 complex which functions as a ubiquitin ligase for the tumor suppressor p53 and which is activated by papillomavirus in cervical cancer (Huang et al., Science 286:1321-26 (1999)). Single subunit ubiquitin ligases having a RING domain include Mdm2, which has also been shown to act as a ubiquitin ligase for p53, as well as Mdm2 itself. Other RING domain, single subunit E3 ligases include: TRAF6, involved in IKK activation; Cbl, which targets insulin and EGF; Sina/Siah, which targets DCC; Itchy, which is involved in haematopoesis (B, T and mast cells); and IAP, involved with inhibitors of apoptosis.
The best characterized E3 ligase is the APC (anaphase promoting complex), which is a multi-subunit complex that is required for both entry into anaphase as well as exit from mitosis (see King et al., Science 274:1652-59 (1996) for review). The APC plays a crucial role in regulating the passage of cells through anaphase by promoting ubiquitin-dependent proteolysis of many proteins. In addition to degrading the mitotic B-type cyclin for inactivation of CDC2 kinase activity, the APC is also required for degradation of other proteins for sister chromatid separation and spindle disassambly. Most proteins known to be degraded by the APC contain a conserved nine amino acid motif known as the xe2x80x9cdestruction boxxe2x80x9d that targets them for ubiquitination and subsequent degradation. However, proteins that are degraded during G1, including G1 cyclins, CDK inhibitors, transcription factors and signaling intermediates, do not contain this conserved amino acid motif. Instead, substrate phosphorylation appears to play an important role in targeting their interaction with an E3 ligase for ubiquitination (see Hershko et al., Ann. Rev. Biochem. 67:429-75 (1998)).
In eukaryotes, a family of complexes with E3 ligase activity play an important role in regulating G1 progression. These complexes, called SCF""s, consist of at least three subunits, SKP1, Cullins (having at least seven family members) and an F-box protein (of which hundreds of species are known) which bind directly to and recruit the substrate to the E3 complex. The combinatorial interactions between the SCF""s and a recently discovered family of RING finger proteins, the ROC/APC11 proteins, have been shown to be the key elements conferring ligase activity to E3 protein complexes. Particular ROC/Cullin combinations can regulate specific cellular pathways, as exemplified by the function of APC11-APC2, involved in the proteolytic control of sister chromatid separation and exit from telophase into G1 in mitosis (see King et al., supra; Koepp et al., Cell 97:431-34 (1999)), and ROC1-Cullin 1, involved in the proteolytic degradation of IxcexaBxcex1 in NF-xcexaB/IxcexaB mediated transcription regulation (Tan et al., Mol. Cell 3(4):527-533 (1999); Laney et al., Cell 97:427-30 (1999)).
Because the E3 complex is the major determinant of selection for protein degradation by the ubiquitin-dependent proteolytic process, modulators of E3 ligase activity may be used to upregulate or downregulate specific molecules involved in cellular signal transduction. Disease processes can be treated by such up- or down regulation of signal transducers to enhance or dampen specific cellular responses. This principle has been used in the design of a number of therapeutics, including Phosphodiesterase inhibitors for airway disease and vascular insufficiency, Kinase inhibitors for malignant transformation and Proteasome inhibitors for inflammatory conditions such as arthritis.
Due to the importance of ubiquitination in cellular regulation and the wide array of different possible components in ubiquitin-dependent proteolysis, there is a need for a fast and simple means for assaying E3 ligase activity. Furthermore, such an assay would be very useful for the identification of modulators of E3 ligase. Accordingly, it is an object of the present invention to provide methods of assaying ubiquitin ligase activity, which methods may further be used to identify modulators of ubiquitin ligase activity.
Tan et al., supra, disclose that ROC1/Cul1 catalyzes ubiquitin polymerization in the absence of target protein substrate. Ohta et al., Mol. Cell 3(4):535-541 (1999) disclose that APC11/APC2 also catalyze ubiquitin polymerization in the absence of target protein substrate, and that this activity is dependent on the inclusion of the proper E2 species. Rolfe et al., U.S. Pat. No. 5,968,761 disclose an assay for identifying inhibitors of ubiquitination of a target regulatory protein.
The present invention provides methods for assaying ubiquitin ligase activity and screening for agents which modulate ubiquitin ligase activity. In one aspect, a method of assaying ubiquitin ligase activity is provided involving the steps of combining ubiquitin, E1, E2 and E3 and measuring the amount of ubiquitin bound to E3. This method may further involve combining a candidate ubiquitin ligase modulator in the combining step. This method does not require a specific target protein to be ubiquitinated. In a preferred embodiment, a substrate protein for ubiquitination other than ubiquitin itself is specifically excluded.
In one embodiment of the assay described above, ubiquitin is in the form of tag1-ubiquitin. In another embodiment, E3 is in the form of tag2-E3. In these embodiments tag1 may be a label or a partner of a binding pair. In one embodiment, tag1 is a fluorescent label, in which case measuring the amount of ubiquitin bound to E3 may be by measuring luminescence.
In another embodiment, tag1 is a member of a binding pair chosen from the group antigen, biotin and CBP. In this latter embodiment, the partner of a binding pair may be labeled by indirect labeling, which may be by a fluorescent label or a label enzyme. The label enzyme may be horseradish peroxidase, alkaline phosphatase or glucose oxidase. When the indirect labeling is by a fluorescent label, measuring the amount of ubiquitin bound to E3 may be by measuring luminescence. In the case that the indirect labeling is by a label enzyme, said enzyme may be reacted with a substrate which produces a fluorescent product, in which case, measuring the amount of ubiquitin bound to E3 may be by measuring luminescence. In one embodiment of the method above, tag1 is a FLAG antigen. In this embodiment, indirect labeling may be by anti-FLAG.
In one aspect of the above method, tag2 is a surface binding molecule, which may be His-tag. In this latter case, the assaying may be performed in a multi-well plate comprising a surface substrate comprising nickel.
In a different embodiment of the method above, when tag1 is a fluorescent label, the combining step further includes combining tag3-ubiquitin. Tag3 may be the second member of a FRET pair with tag1 or it may be a quencher of tag1. In this embodiment, measuring the amount of ubiquitin bound to E3 may be by measuring fluorescent emission, which may involve measuring the fluorescent emission spectrum. In this last embodiment, the method may further comprise comparing the measured fluorescent emission spectrum with the fluorescent emission spectrum of unbound tag1- and tag3-ubiquitin. When measuring the amount of ubiquitin bound to E3 is by measuring the fluorescent emission spectrum, this measuring may be continuous or at specific time points following the original combining of materials.
In another aspect of the invention, a method of identifying modulators of ubiquitination enzymes is provided. This method involves combining tag1-ubiquitin, a candidate modulator, E1, E2 and tag2-E3 and measuring the amount of tag1-ubiquitin bound to tag2-E3. In another embodiment, this method further comprises combining tag1-ubiquitin, a candidate modulator, E1 and tag2-E2 and measuring the amount of tag1-ubiquitin bound to the tag2-E2. In a preferred embodiment, target protein (i.e., a substrate protein other than ubiquitin itself) is specifically excluded in the method.
In the embodiments of the method of identifying modulators of ubiquitination enzymes , tag1 may be a label or a partner of a binding pair. If tag1 is a label, it may be a fluorescent label, in which case, measuring the amount of bound tag1-ubiquitin may be by measuring luminescence. If tag1 is a partner of a binding pair, the potential binding pair partners, labeling options and subsequent measuring options are substantially as described for tag1 above for the method of assaying ubiquitin ligase activity.
In the above method of identifying modulators of ubiquitination enzymes, tag2 and tag3 may be surface substrate binding molecules. Options for such molecules and conditions for performing the method are as described for the method of assaying ubiquitin ligase activity.
In another aspect of the invention, a method of assaying ubiquitination enzyme activity is provided. This method comprises combining tag1-ubiquitin and tag2-ubiquitin, E1, E2 and E3 under conditions in which ubiquitination can take place and measuring the amount or rate of ubiquitination. In this embodiment, tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label and tag2 is a quencher of tag1. In one embodiment, the method includes combining a candidate ubiquitination modulator with the other components. In a preferred embodiment of this method, measuring is by measuring the fluorescent emission spectrum from the combination, preferably continuously or at specific time points following combining the components. These measurements may be compared to the fluorescent emission spectrum of unbound tag1 and tag2 ubiquitin.
Also provided herein is a method of identifying a ubiquitination modulator. This method involved combining a candidate ubiquitination modulator, tag1-ubiquitin and tag2-ubiquitin, E1, E2 and E3 under conditions in which ubiquitination can take place and measuring the amount or rate of ubiquitination. In this embodiment, tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label and tag2 is a quencher of tag1. In one embodiment, the method includes combining a candidate ubiquitination modulator with the other components. In a preferred embodiment of this method, measuring is by measuring the fluorescent emission spectrum from the combination, preferably continuously or at specific time points following combining the components. These measurements may be compared to the fluorescent emission spectrum of unbound tag1 and tag2 ubiquitin.
In the latter two assays described, the ubiquitin may be in the form tag1,3-ubiquitin and tag2,3-ubiquitin, wherein tag3 is a member of a binding pair, preferably FLAG. In another embodiment of these assays, E3 may be in the form of tag4-E3, wherein tag4 is a surface substrate bonding molecule.
In still another aspect of the invention, compositions are provided for use in assaying ubiquitination. The composition comprises tag1-ubiquitin and tag2-ubiquitin, wherein tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label and tag2 is a quencher of tag1. In one embodiment, the composition further comprises E1, E2 and E3. In a preferred embodiment, the composition further comprises a candidate ubiquitination modulator. In yet another embodiment, the composition comprises a target protein.
In addition, provided herein are compositions for use in assaying for a ubiquitination modulator. The composition comprises a candidate ubiquitination modulator, tag1-ubiquitin and tag2-ubiquitin, wherein tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent label and tag2 is a quencher of tag1. In one embodiment, the composition further comprises E1, E2 and E3. In one embodiment, the composition comprises a target protein.
In preferred embodiments of the assays and compositions described above, E2 is selected from the group consisting of Ubc5, Ubc3, Ubc4 and UbcX. In ap referred embodiment, E3 comprises a RING finger protein, preferably selected from the group consisting of ROC1, ROC2 and APC 11. In a preferred embodiment, E3 comprises a Cullin, preferably selected from the group consisting of CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and APC2. In a preferred embodiment, E3 comprises a RING finger protein/Cullin combination, preferably selected from the group consisting of APC11/APC2, ROC1/CUL1, ROC1/CUL2 and ROC2/CUL5.
Other aspects of the invention will become apparent to the skilled artisan from the following description of the invention.